Stringent mating-type-regulated auxotrophy increases the accuracy of systematic genetic interaction screens with Saccharomyces cerevisiae mutant arrays.
A genomic collection of haploid Saccharomyces cerevisiae deletion strains provides a unique resource for systematic analysis of gene interactions. Double-mutant haploid strains can be constructed by the synthetic genetic array (SGA) method, wherein a query mutation is introduced by mating to mutant arrays, selection of diploid double mutants, induction of meiosis, and selection of recombinant haploid double-mutant progeny. The mechanism of haploid selection is mating-type-regulated auxotrophy (MRA), by which prototrophy is restricted to a particular haploid genotype generated only as a result of meiosis. MRA escape leads to false-negative genetic interaction results because postmeiotic haploids that are supposed to be under negative selection instead proliferate and mate, forming diploids that are heterozygous at interacting loci, masking phenotypes that would be observed in a pure haploid double-mutant culture. This work identified factors that reduce MRA escape, including insertion of terminator and repressor sequences upstream of the MRA cassette, deletion of silent mating-type loci, and utilization of alpha-type instead of a-type MRA. Modifications engineered to reduce haploid MRA escape reduced false negative results in SGA-type analysis, resulting in >95% sensitivity for detecting gene-gene interactions.
Singh
Indira
I
Department of Genetics, University of Alabama, Birmingham, Alabama 35294, USA.
Pass
Rebecca
R
Togay
Sine Ozmen
SO
Rodgers
John W
JW
Hartman
John L
JL
D050539:3-Isopropylmalate Dehydrogenase / Q000235:genetics; D001483:Base Sequence; D002172:Canavanine / Q000494:pharmacology; D015825:Chromosomes, Fungal / Q000235:genetics; D015169:Colony Count, Microbial; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D004171:Diploidy; D017353:Gene Deletion; D049770:Genes, Mating Type, Fungal; D005821:Genetic Techniques; D006238:Haploidy; D006579:Heterozygote; D008540:Meiosis / Q000187:drug effects; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012641:Selection, Genetic; D013728:Terminator Regions, Genetic / Q000235:genetics
D029701:Saccharomyces cerevisiae Proteins; C000204:S-2-aminoethyl cysteine; D002172:Canavanine; D050539:3-Isopropylmalate Dehydrogenase; C524993:LEU2 protein, S cerevisiae; D003545:Cysteine